Method For The Analysis Of Point Mutations

Abstract

The invention relates to a method for determining point mutations by means of DNA microarrays and TIRF excitation. According to said method, bonding of nucleic acids to short DNA probes on a microarray is measured at different temperatures. Melting point curves are generated from the measured values and the difference in the melting point curves between the probe for the wild-type DNA and the probe for the corresponding mutated DNA is generated. The position of said curves makes it possible to unambiguously decide whether the point mutation is a homozygous DNA or a heterozygous DNA and what type of homozygosity it is.

1 . A method for the analysis of nucleic acids, comprising: 
 (a) measurement of the melting curve of a nucleic acid hybrid of a sample nucleic acid with a first nucleic acid probe,    (b) measurement of the melting curve of a nucleic acid hybrid of a sample nucleic acid with a second nucleic acid probe, which differs from said first nucleic acid probe by at least one nucleotide, but which is at least 80% homologous with said first nucleic acid probe,    (c) mathematical correlation of the melting curve from (b) with the melting curve from (a), and    (d) correlation of the result from (c) with reference values for the possible compositions of the nucleic acid samples.    
     
     
         2 . The method of  claim 1  comprising the further step of 
 (e) confirmation of a finding on the basis of the collected data.    
     
     
         3 . A method for the determination of the identity of a nucleic acid, wherein: 
 (a) the melting point curve of the bond of a reference nucleic acid of an identical sequence is recorded on a nucleic acid probe,    (b) the melting point curve of the nucleic acid to be examined is recorded on a nucleic acid probe of an identical sequence, and    (c) both of the melting point curves are mathematically correlated.    
     
     
         4 . The method according to  claim 1 , wherein the data are acquired by means of a heatable TIRF apparatus.  
     
     
         5 . The method according to  claim 1 , wherein said nucleic acid probes are covalently bonded to the surface of a waveguide.  
     
     
         6 . The method according to  claim 1 , wherein said nucleic acid probes are covalently bonded to a polymer or a polymer layer.  
     
     
         7 . The method according to  claim 5 , wherein a plurality of nucleic acid probes is attached to the waveguide chip.  
     
     
         8 . The method according to  claim 5 , wherein said waveguide chip is freed from the bonded nucleic acids after the performance of an experiment and used for at least one other experiment.  
     
     
         9 . The method according to  claim 5 , wherein said waveguide chip is hybridized with a reference nucleic acid, in order to obtain correlation values for the subsequent measurements.  
     
     
         10 . A method for the reduction of unspecific signals on DNA chips, wherein, 
 (a) a DNA chip with an unlabeled sample, which bonds on said nucleic acid probes on said chip, is hybridized,    (b) the hybridization is then dissolved, and    (c) said DNA chip is then used in at least one other hybridization experiment with labeled DNA.    
     
     
         11 . The method of  claim 3 , wherein the data are acquired by means of a heatable TIRF apparatus.  
     
     
         12 . The method of  claim 3 , wherein said nucleic acid probes are covalently bonded to a surface of a waveguide.  
     
     
         13 . The method of  claim 3 , wherein said nucleic acid probes are covalently bonded to a polymer or a polymer layer.  
     
     
         14 . The method of  claim 12 , wherein a plurality of nucleic acid probes is attached to the waveguide chip.  
     
     
         15 . The method of  claim 12 , wherein said waveguide chip is freed from the bonded nucleic acids after the performance of an experiment and used for at least one other experiment.  
     
     
         16 . The method of  claim 12 , wherein said waveguide chip is hybridized with a reference nucleic acid, in order to obtain correlation values for the subsequent measurements.